Capture compositions

ABSTRACT

Disclosed is a covalently-linked multilayered three-dimensional matrix comprising capture molecules, linkers and spacers (referred to as a Molecular Net) for specific and sensitive analyte capture from a sample. Also disclosed herein is a Molecular Net comprising covalently-linked multilayered three-dimensional matrix comprising more than one type of capture molecule and more than one type of linker and may comprise one or more spacer for specific and sensitive capture of more than one type of analyte from a sample. A Molecular Net may comprise a pseudorandom nature. Use of various capture molecules, linkers and spacers in a Molecular Net may confer unique binding properties to a Molecular Net. Porosity, binding affinity, size exclusion abilities, filtration abilities, concentration abilities and signal amplification abilities of a Molecular Net may be varied and depend on the nature of components used in its fabrication. Uses of a Molecular Net may include analyte capture, analyte enrichment, analyte purification, analyte detection, analyte measurement and analyte delivery. Molecular Nets may be used in liquid phase or on solid phases such as nanomaterials, modified metal surfaces, nanospheres, microspheres, microtiter plates, slides, pipettes, cassettes, cartridges, discs, probes, lateral flow devices, microfluidics devices, microfluidics devices, optical fibers and others.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/642,393 filed on Jul. 6, 2017 (which is herein incorporated byreference in its entirety), which is a continuation of U.S. applicationSer. No. 14/214,556 filed on Mar. 14, 2014 (which is herein incorporatedby reference in its entirety), which claims the benefit U.S. ProvisionalApplication No. 61/783,189, filed on Mar. 14, 2013 (which is hereinincorporated by reference in its entirety). U.S. application Ser. No.14/214,556 filed on Mar. 14, 2014 is a continuation-in-part of U.S.application Ser. No. 13/511,364 with a 371(c) date of Oct. 7, 2012(which is herein incorporated by reference in its entirety). U.S.application Ser. No. 14/214,556 filed on Mar. 14, 2014 is acontinuation-in-part of U.S. application Ser. No. 13/938,055, filed Jul.9, 2013 (which is herein incorporated by reference in its entirety).

U.S. application Ser. No. 15/642,393 filed on Jul. 6, 2017 is also acontinuation-in-part of U.S. application Ser. No. 13/938,055 filed onJul. 9, 2013 (which is herein incorporated by reference in itsentirety), which claims the benefit of U.S. Provisional Application No.61/699,261, filed on Jul. 9, 2012 (which is herein incorporated byreference in its entirety) and which claims the benefit of U.S.Provisional Application No. 61/669,265, filed on Jul. 9, 2012 (which isherein incorporated by reference in its entirety). U.S. application Ser.No. 13/938,055 filed on Jul. 9, 2013 is a continuation-in-part of U.S.application Ser. No. 13/511,364 with a 371(c) date of Oct. 7, 2012.

U.S. application Ser. No. 15/642,393 filed on Jul. 6, 2017 is also acontinuation-in-part of U.S. application Ser. No. 13/511,364 with a371(c) date of Oct. 7, 2012 (which is herein incorporated by referencein its entirety) which is a 371 national stage entry application ofPCT/US10/58086 filed on Nov. 24, 2010 (which is herein incorporated byreference in its entirety), which (a) claims the benefit of U.S.Provisional Application No. 61/410,837, filed on Nov. 5, 2010 (which isherein incorporated by reference in its entirety), (b) claims thebenefit of U.S. Provisional Application No. 61/343,467, filed on Apr.29, 2010 (which is herein incorporated by reference in its entirety),(c) claims the benefit of U.S. Provisional Application No. 61/340,287,filed on Mar. 15, 2010 (which is herein incorporated by reference in itsentirety), (d) claims the benefit of U.S. Provisional Application No.61/337,257, filed on Feb. 1, 2010 (which is herein incorporated byreference in its entirety), and (e) claims the benefit of U.S.Provisional Application No. 61/281,991, filed on Nov. 24, 2009 (which isherein incorporated by reference in its entirety).

BACKGROUND

Current strategies for solid phase analyte capture, analyte detectionand analyte measurement exist using a single layer of capture moleculesabsorbed or covalently tethered to a surface for direct real-timesensing or are used in conjunction with secondary detection steps in anindirect detection modality are well known in the art. Both direct andindirect methods have demonstrated limitations in sensitivity,specificity, signal-to-noise ratio and/or cost.

There is need for analyte capture technology for solid phase surfaces ordevices that can selectively capture analytes from a complex sample withlittle or no sample preparation and to position said selected analytesin a manner to maximize captured analyte measurement and/or detection ina manner that is compatible with most technologies.

SUMMARY

Devices for capturing an analyte are described. In one embodiment, adevice may comprise a solid phase and a molecular net coupled to atleast a portion of a surface of the solid phase. The molecular net mayinclude capture molecules of at least one type coupled to each other bylinker molecules of a plurality of types to form a covalently-linkedmulti-layered three-dimensional matrix. The capture molecules may beconfigured to bind to the analyte.

Methods of manufacturing a device for capturing an analyte are alsodescribed. In one embodiment, a method may comprise providing a solidphase, and placing a molecular net on at least a portion of a surface ofthe solid phase. The molecular net may include capture molecules of atleast one type coupled to each other by linker molecules of a pluralityof types to form a covalently-linked multi-layered three-dimensionalmatrix. The capture molecules may be configured to bind to the analyte.

Methods of measuring a quantity of an analyte in a sample are alsodescribed. In one embodiment, a method may comprise providing one ormore devices each comprising a solid phase and a molecular net coveringat least a portion of a surface of the solid phase. The molecular netmay include capture molecules of at least one type coupled to each otherby linker molecules of a plurality of types to form a covalently-linkedmulti-layered three-dimensional matrix. The capture molecules configuredto bind to the analyte. The method also comprises exposing the devicesto the sample and allowing at least a portion of the analyte to bind tothe capture molecules of the molecular nets of the devices.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of traditionally conjugated microparticles andMolecular Net microparticles in IgG purification.

FIG. 2 shows a traditional capture molecule conjugation tomicroparticles and the corresponding analyte measurement capability.

FIG. 3 shows an effectiveness of Molecular Net microparticles inmeasuring analyte.

FIG. 4 shows an effectiveness of a Molecular Net with topology inmeasuring analyte.

FIG. 5 shows a comparison of traditionally conjugated microparticles andMolecular Net microparticles in a Tau ELISA.

FIG. 6 shows a comparison of traditionally conjugated microparticles andMolecular Net microparticles in a TSH Luminex sandwich immunoassay.

FIG. 7 shows an exemplary Molecular Nets on particles.

FIG. 8 shows an exemplary Molecular Net topological features onparticles.

FIG. 9 shows an exemplary Molecular Nets for analyte delivery.

FIG. 10 shows examples of Molecular Nets for analyte purification from asample.

FIG. 11 shows examples of Molecular Nets for analyte detection andmeasurement from a sample.

DESCRIPTION

It has been shown in U.S. patent serial nos. 61/281,991, 61/337,257,61/340,287, 61/343,467, 61/410,837, 61/489,646, and 61/489,648, each ofwhich are hereby incorporated by reference, that the construction anduse of a covalently-linked pseudorandom multilayered three-dimensionalmatrix enables the rapid and specific capture of protein, nucleic acid,carbohydrate, lipid, cell or other analytes from an unprocessed sampleand that the use of such Molecular Net may be a significant improvementupon conventional analyte binding approaches.

Design and Fabrication of Molecular Nets

Properties of Molecular Nets may be imparted by: the capture moleculesselected for use (examples of capture molecules may include antibodies,nucleic acid probes, enzymes, recombinant proteins, peptides andothers); the resultant specificity said capture molecules impart; thesize and number of selected capture molecules; the placement and spacingof the capture molecules in the molecular Net layer(s); the combinationof capture molecules; the order in which the capture molecules may beused; and the ratio of capture molecules to linker molecules and spacermolecules used.

Properties of Molecular Nets may also be imparted by: the linkermolecules selected for use (examples of linker molecules includehomobifunctional, heterobifunctional, trifunctional and multifunctionaltypes); the chemical specificity of the linker molecules; the Angstromlength of the linker molecules; the combination of linker molecules; theorder in which the linker molecules may be used; and the ratio ofcapture molecules to linker molecules and spacer molecules used.

Properties of Molecular Nets may also be imparted by: the spacermolecules selected for use (examples of spacer molecules include PEG,polymer, nucleic acid, albumin, Fc region, peptide, and other); thechemical properties of the spacer molecules; the size and number ofspacer molecules; the order in which the spacer molecules may be used;and the ratio of spacer molecules to linker molecules and capturemolecules used.

Placement and spacing of the capture molecules, linker molecules andspacer molecules may: confer a characteristic topology on the MolecularNet surface; confer a characteristic density within each layer of aMolecular Net; confer a characteristic porosity of a Molecular Net;remove spatial constraints and thus stearic hindrance; improve bindingcapacity; reduce non-specific binding; enable the binding of multipleforms of analyte (for example, simultaneous capture of degraded analyte,whole analyte and complexed analyte), and other.

The porosity within a Molecular Net may be random, pseudorandom orirregularly interspersed. Porosity of a Molecular Net may be used tofilter a sample; may be used to discriminate binding potential moleculesin a sample by size-exclusion; may be used to enable macromolecular orcellular binding due to the reduction in stearic hindrance, or other.The pores of a Molecular Net comprise capture molecules, linkermolecules and may comprise spacer molecules. Traditional approaches togenerate porosity on a solid phase relates to the mechanicalmodification of the surface of the solid phase and employ methods suchas laser etching, laminating, lithography, laser printing or others togenerate pores, holes or other structures in the solid surface. Thissolid surface is then prepared for accepting conjugating capturemolecules. Use of a Molecular Net removes the need for mechanicalmodification of a surface and is thus more cost-effective. Additionally,traditional approaches are still hampered by the problem of highnon-specific binding and require capture chemistry to be bound to themechanically modified solid phase, which is not an improvement.Additionally, flexibility may be imparted into a Molecular Net ascompared to the traditional capture format due to size-exclusionproperties conferred by the porosity built into each layer of aMolecular Net. In some layers, pore diameter and depth may be similar ormay vary depending on the application. In some layers, pore sizes mayvary, the variance of which may depend on the application.

Porosity that may be imparted on a Molecular Net may include but are notlimited to picopores, nanopores, micropores, filtration pores, sievingpores, pockets or other. Porosity may be imparted into a Molecular Netby the selection of and method of incorporation of specific capturemolecules, linker molecules and spacer molecules into each layer of aMolecular Net. Porosity may also be imparted into a Molecular Net by theselection of and method of incorporation of specific capture molecules,linker molecules and spacer molecules used in the fabrication ofsequential layers.

Molecular Net porosity may range from about 6 Angstroms in diameter tomore than about 1 um in diameter based on the identity of capturemolecules, linker and spacers used in a layer. In some cases, theporosity of a Molecular Net may comprise a range of pore diameters.Exemplary diameter ranges may be from about 5 nm to about 50 nm, fromabout 10 nm to about 100 nm, from about 50 nm to about 200 nm, fromabout 250 nm to about 500 nm, from about 500 nm to about 1 um, and fromabout 800 nm to about 1.5 um.

In some cases, capture molecules may be used to generate pores in aMolecular Net. In these instances, capture molecules may be pre-linkedto one another prior to being incorporated into a Molecular Net layer.In some cases, linkers may be selected based on Angstrom length of thespacer arms. In some examples, extenders may be used to connect a firstlinker to a second linker to generate a long multi-functional linker. Insome cases, spacers may be used to generate pores in a Molecular Net.Spacers may also be pre-linked to one another prior to beingincorporated into a Molecular Net layer. In other examples, inertphysical plugs may be used to build a pore, whereby each physical plugmay be placed on a previously built layer while a new layer is beingconstructed. After curing, the physical plugs may be removed, thusleaving a pore of a specific diameter.

The flexible nature of the Molecular Net enables the use of multipletypes of capture molecules. In some examples, a Molecular Net comprisesa single type of capture molecule. In other examples, a Molecular Netcomprises multiple types of capture molecules. In some examples, the useof more than one monoclonal antibody during the fabrication of aMolecular Net enables said Molecular Net to bind more than one epitopeof an analyte. Use of more than one type of epitope-specific capturemolecule enables improved analyte capture by a Molecular Net and relatesto its performance. In some examples, the use of more than one nucleicacid sequence may be used during the fabrication process to generate aMolecular Net capable of binding to more than one epitope of an analyte.Examples of benefit depend on the use of the Molecular Net and maycomprise improved performance in terms of minimum levels of detection,sensitivity, positive predictive value, negative predictive value,ability to work with degraded samples, ability to work with a diversepopulation, and others when used in a test; may comprise improvedperformance in binding capacity, purity, binding kinetics, targetanalyte depletion, and others when used as a purification tool; orother.

In some examples, the use of capture molecules directed againstmutually-confirmatory analytes may be used in a Molecular Net andrelates to its performance. Use of mutually-confirmatory capturemolecules in a Molecular Net may be used in a confirmatory manner,whereby the capture of more than one analyte may provide a morestatistically significant positive result; may provide a more robusttest result; may provide additional information regarding a sample; andother. Use of mutually-confirmatory capture molecules in a Molecular Netmay also be used to qualify a sample or may be used as a control in atest or may be used to measure more than one related molecular variablelinked to a disease state or may be used to measure more than onerelated molecular variable linked to the treatment of a disease.

Examples of mutually-confirmatory analytes a Molecular Net may befabricated to simultaneously capture from a sample may include: geneticsequence and corresponding protein product (for examples, cancer-relatedSNPs in BRCA1 and BRCA1 protein); the mRNA and corresponding proteinproduct (for example, human lactase mRNA and Lactase protein); thegenetic sequence and the corresponding mRNA product (for example,disease-related SNPs in LMNA and pre-spliced or spliced Lamin A/C mRNA);miRNA and related mRNA or protein products (miR 9 and REST or CoRESTmRNA, or miR 9 and REST protein); small molecule drugs and drug targets(tofacitinib and Janus kinase 3); epitope-specific biologics and therespective targets (for example, anti-TNF antibodies and circulating TNFcytokine); epitope-specific antibodies, epitope-specific T cells and/orepitope-specific B cells or the like (for example, anti-DNAautoantibodies, anti-DNA CD4⁺ T cells and/or anti-DNA B cells); orothers. Examples of benefit depend on use and may relate to improvedperformance in test sensitivity, positive predictive value, negativepredictive value, specificity, diagnosis of a disease, ability to workwith samples experiencing genetic drift, ability to measure response toa therapeutic, ability to measure effectiveness of a therapeutic, orother.

In one example, a Molecular Net may be fabricated in a manner to captureand position bound analytes in a manner that enhances the intensity of adetectable signal or may enhance detection of bound analytes, such aswhen used in a test with optical detection. Placement of capturedanalytes in a layered manner by pre-positioned layered capture moleculesmay enable the rapid detection of analyte by signal intensification.Examples of signal intensification by a Molecular Net may relate tofluorescence, fluorescence resonance energy transfer, absorbance,luminescence, light scatter, surface plasmon resonance, opticalheterodyne detection, or other.

Said Molecular Net may be designed and fabricated to replace the needfor costly and time-intensive methods for ultra-sensitive detection suchas PCR, branched DNA, or multi-step detection methods required forsignal amplification. Said Molecular Net may also be designed andfabricated to replace the need for costly and complicated analyticaldevices.

Generally, the number of capture molecules incorporated into a3-dimensional Molecular Net matrix is less than or equivalent to thenumber of capture molecules conjugated in a 2-dimensional manner to asurface using conventional approaches. Two-dimensional capturemolecule-surface conjugates may rely on the use of a single linker typeor may rely on the sequential use of 2 linkers to conjugate capturemolecules to a solid surface. During the fabrication of a layer of aMolecular Net, multiple linker types are used simultaneously to linkcapture molecule to capture molecule of a new layer and the linkedcapture molecules of a new layer to a spacer or capture molecule of aprevious layer. Molecular Nets may be fabricated in solution prior toplacement on a solid surface. Pre-fabricated Molecular Nets may beabsorbed or covalently linked to a solid surface. Molecular Nets mayalso be fabricated directly onto a solid surface, layer by layer. SaidMolecular Nets may be placed on a solid surface using non-covalent(electrostatic, van der Waals, or other) or covalent methods. In someinstances, polystyrene, polyurethane, polyethylene or treated surfacessuch as poly-L-lysine coated surfaces, modified surfaces comprising—COOH, NHS, amine or other may be purchased from commercial sources(examples of vendors may include Thermo, Millipore, Luminex and other)and used as solid phase surfaces for Molecular Net placement. In otherinstances, solid phase surfaces may be pre-treated by chemicals such asacid to activate the surface moieties and thus to generate attachmentpoints between the solid phase surface and reactive moieties of aMolecular Net. In some examples, a solid phase may be pre-treated withlinker to covalently link a solid phase surface to a Molecular Net.

Design and fabrication of a Molecular Net for use on a solid phasesurface may result in a covalently-linked multilayered three-dimensionalmatrix of capture molecules secured by covalent connectors within eachlayer. Design and fabrication may occur in a sequential manner where afirst layer is fabricated and subsequent layers are fabricated in asequential manner whereby each layer may be interconnected in a covalentmanner to enhance structural integrity, topology, porosity and/orstability. Selection of individual capture molecules, linkers andspacers may be made to contribute to one or more property of a MolecularNet. Properties may comprise analyte specificity, thermal stability,layer thickness, pore diameter, absorbance spectra, emission spectra,solid phase compatibility or other.

The use of capture molecules and linker molecules and spacers of knownlengths and widths may be used to generate various topology on thesurface of the Molecular Net. Topological features that may be impartedon a Molecular Net may include but are not limited to dimples, pocks,stipples, pores, mounds, branches, filaments, fibers, fissures, raisedsegments or other and may be arranged in a Molecular Net in a random,pseudorandom or irregular manner.

Topological features of a Molecular Net may be generated through the useof capture molecules and linkers; capture molecules, linkers andspacers; or linkers and spacers. In some cases, capture molecules may beused to generate topological features of a Molecular Net. In theseinstances, capture molecules may be pre-linked to one another prior tobeing incorporated covalently into a Molecular Net layer. In some cases,linkers may be selected based on Angstrom length of the spacer arms. Insome examples, spacers may be used to connect a first linker to a secondlinker to generate a long multi-functional linker. In some cases,spacers may be used to generate topological in a Molecular Net. Spacersmay also be pre-linked to one another or to capture molecules prior tobeing incorporated into a Molecular Net layer.

Molecular nets may be designed and fabricated to impart characteristicssuch as affinity, size exclusion, filtration, fluorescence, and otherinto each layer of a Molecular Net. Specific capture molecules, linkermolecules and spacer molecules may be selected based on size, length,diameter, thickness, optical properties, chemical properties or otherfor imparting characteristics into a Molecular Net during thefabrication process.

Molecular Nets may be fabricated in a manner whereby one or more capturemolecules may serve a structural role, may serve both a structural roleand a role in analyte capture within the covalently-linked multilayeredthree-dimensional matrix. Some examples of capture molecules that may beused for structural and/or analyte capture roles in a Molecular Net.

The distance between capture molecules in each layer of a Molecular Netmay be determined, in part, by the diameter, width and/or length ofcapture molecules, linkers and spacers used in the fabrication processfor each layer, whereby the molar relationship between eachlinker-capture-spacer molecule may be similar or may be different andthe selection of said molecules may be dependent on size and/or shape ofthe analyte to be captured, the method used to measure captured analyteand/or desired use.

Molecular Nets may be designed and fabricated in a manner whereby eachcapture molecule, linker and spacer component may have equivalent ornon-equivalent molar ratios in a layer of said Molecular Net. Varianceof molar ratios between said components may be used from time to time togenerate porosities or other topological features within each layer.Said porosities and topological features may have a range of diametersand may have a range of associated depths. Variance of molar ratiosbetween Molecular Net components may occur in a single layer ofMolecular Net or may occur in more than one layer of a Molecular Net andis dependent on the intended use of a Molecular Net.

TABLE 1 Examples of Molecular Net structural components with analytecapture ability. Approximate Approximate Examples of Molecular NetDiameter Length Structural/Capture Components (nm) (nm) IgG, IgE ~9 16IgM 37 37 IgA ~9 ~32 Streptavidin & recombinant variants ~105(tetrameric) N/A Protein A & recombinant variants ~3.2-5.3  N/A ProteinG & recombinant variants  ~3-5.4 N/A MHC I 3.05 N/A MHC II 2.99 N/A TCR3.34 N/A CD28 2.75 N/A TLR 4 2.62 N/A B7x 4.52 N/A Taq polymerase ~6.49N/A poly(Arg₉) peptide 1.43 N/A HSP70 3.46 N/A

Some examples of analyte dimensions that may be considered during thedesign and fabrication process are provided in Table 2. Design andfabrication of Molecular Net surface chemistry, pore diameter, topology,layering or other may be based on analyte shape; analyte structure,analyte isoforms, analyte charge, analyte complex formation with othermolecules, and other forms. Furthermore, Molecular Nets may be designedand fabricated to bind and capture said analyte or may be designed andfabricated to exclude said analyte. Examples of analytes and analytesizes can be found in Table 2.

TABLE 2 Examples of analytes and their dimensions. ApproximateApproximate Exemplary Diameter Length Analyte (um) (um) E. coli 0.5 1-2Klebsiella spp. 0.3-1   0.6-6   Pseudomonas spp. 0.6 3 Staphylococcus 11 aureus Staphylococcus >10 >10 aureus (cluster) Enterotoxin K ~4.29 N/APeptidoglycan, gram negative ~2-3, species Highly bacteria dependentspecies dependent Outer membrane, gram negative ~7, species Speciesbacteria dependent dependent IgG, IgE 0.009 0.016 IgA 0.009 0.032 IgM0.037 0.037 B cell - G0 phase of cell cycle 4.5-5.5 N/A B cell - earlyG1 phase of cell 5.5-7   N/A cycle B cell - late G1 and S phase of  7-10N/A cell cycle B cell - late S, G2 and M phases 10-12 N/A of cell cycleMonocyte ~9-18 N/A Macrophage 21, activation N/A level dependentNeutrophil 7.17-9.3, N/A activation level dependent IL6 monomer ~4.11N/A IL6 multimer (variable) ~6.16 N/A IL10 monomer ~3.88 N/A IL10multimer (variable) ~7.7 N/A microRNA-146a ~3-6  ~7-9 

Molecular Nets comprising structural components and capture componentsmay be arranged in the covalently linked 3-dimensional (3D) multilayeredmatrix and may relate to the capture of one or more analyte relating toone or more of the following characteristics: surface chemistry; analyteshape; analyte structure; analyte isoforms; analyte charge;post-translational modification; chemical modification; activity; orother.

Molecular Nets may comprise structural components that also act in amanner relating to the capture of analytes and may be arranged in theinterconnected 3D multilayered matrix of a Molecular Net by covalentlinkers. A Molecular Net may also comprise spacers to interconnect saidstructure/capture molecules in a manner to maximize structuralreinforcement, stability and/or specific analyte capture capability.Molecular Net examples comprising capture components/structuralcomponents, linkers and spacers are presented in Table 3.

Fabrication of the molecular Net is unique in that capture molecules aresecured in a 3D matrix by covalent linker molecules. In numerousstudies, Molecular Nets have been demonstrated to have improved thermalstability and extend shelf-life beyond traditional capture technologies.

TABLE 3 Examples of Molecular Nets and their Use. Capture Molecules toMethods of Analyte to Use for Affinity Generating Size AnticipatedCapture Capture Exclusion Use E. coli Antibodies against Covalentlylinked Diagnostics: Food surface antigens (e.g., antibodies - IgG,safety, infectious LPS, O-antigen, pili, IgM, covalent disease, watersafety; other); PNA probes linkers, spacers Molecular tools: againstchromosomal polymicrobial and/or plasmid DNA sampling, microbiomesampling, molecular biology Klebsiella spp. Antibodies againstCovalently linked Diagnostics: Food surface antigens (e.g., antibodies -IgG, safety, infectious LPS, other); PNA IgM, covalent disease, watersafety; probes against linkers, spacers Molecular tools: chromosomaland/or polymicrobial plasmid DNA sampling, microbiome sampling,molecular biology Pseudomonas spp. Antibodies against Covalently linkedDiagnostics: Food surface antigens (e.g., antibodies - IgG, safety,infectious LPS, V antigen, other), IgM, covalent disease, water safety;excreted materials linkers, spacers Molecular tools: (e.g., heat shockpolymicrobial proteins, alginate, sampling, microbiome other); PNAprobes sampling, molecular against chromosomal biology and/or plasmidDNA Staphylococcus Antibodies against Covalently linked Diagnostics:Food aureus surface antigens (e.g., antibodies - IgG, safety, infectiousprotein A, IgM, covalent disease, water safety; peptidoglycan, other),linkers, spacers Molecular tools: excreted materials polymicrobial(e.g., heat shock sampling, microbiome proteins, exotoxins, sampling,molecular other); PNA probes biology against chromosomal and/or plasmidDNA Staphylococcus Antibodies against Covalently linked Diagnostics:Food aureus (cluster) surface antigens (e.g., antibodies - IgG, safety,infectious protein A, IgM, covalent disease, water safety;peptidoglycan, other), linkers, longer Molecular tools: excretedmaterials spacers polymicrobial (e.g., heat shock sampling, microbiomeproteins, exotoxins, sampling, molecular other); PNA probes biologyagainst chromosomal and/or plasmid DNA IgG, IgE Antibodies against FcCovalently linked Diagnostics: Immune IgG or IgE; Antibodies antibodies,response profiling, against Fab; antigens antigens, covalentvaccination, antibody linkers, spacers titering; Molecular tools:immunologic studies, pre-clinical studies IgA Antibodies against FcCovalently linked Diagnostics: Immune IgA; Antibodies antibodies,response profiling, against Fab IgA; antigens, covalent vaccination,antibody antigens linkers, spacers titering; Molecular tools:immunologic studies, pre-clinical studies IgM Antibodies againstCovalently linked Diagnostics: Immune IgM; Antibodies antibodies,response profiling, against 5□ IgM; antigens, covalent vaccination,antibody antigens linkers, spacers titering; Molecular tools:immunologic studies, pre-clinical studies B cell - G0 phase Antibodiesagainst Covalently linked Diagnostics: Immune of cell cycle PAX5, CD19,CD20, antibodies - IgG, response profiling, CD79a, others; IgM, covalentdisease monitoring; antigens; TCR: antigen; linkers, spacers, Moleculartools: MHC I: antigen; MCH MHC: antigen immunologic studies, II:antigen; cytokines complexes, pre-clinical studies (e.g., IL10, IL6,TGFb, cytokines other) B cell - early G1 Antibodies against Covalentlylinked Diagnostics: Immune phase of cell cycle PAX5, CD19, CD20,antibodies - IgG, response profiling, CD79a, others; IgM, covalentdisease monitoring; antigens; TCR: antigen; linkers, spacers, Moleculartools: MHC I: antigen; MCH MHC: antigen immunologic studies, II:antigen; cytokines complexes, pre-clinical studies (e.g., IL10, IL6,TGFb, cytokines other) B cell - late G1 and Antibodies againstCovalently linked Diagnostics: Immune S phase of cell PAX5, CD19, CD20,antibodies - IgG, response profiling, cycle CD79a, others; IgM, covalentdisease monitoring; antigens; TCR: antigen; linkers, spacers, Moleculartools: MHC I: antigen; MCH MHC: antigen immunologic studies, II:antigen; cytokines complexes, pre-clinical studies (e.g., IL10, IL6,TGFb, cytokines other) B cell - late S, G2 Antibodies against Covalentlylinked Diagnostics: Immune and M phases of PAX5, CD19, CD20,antibodies - IgG, response profiling, cell cycle CD79a, others; IgM,covalent disease monitoring; antigens; TCR: antigen; linkers, spacers,Molecular tools: MHC I: antigen; MCH MHC: antigen immunologic studies,II: antigen; cytokines complexes, pre-clinical studies (e.g., IL10, IL6,TGFb, cytokines other) Macrophage Complement, Covalently linkedDiagnostics: Immune antibodies against antibodies - IgG, responseprofiling, mannose receptor Ab, IgM, covalent infectious diseaseanti-Ly6C, or other; linkers, spacers, monitoring; antibodies againstM1, MHC: antigen vaccination M2a, M2b, M2c complexes, monitoring;chronic markers; TLR agonists; cytokines, TLR inflammatory diseasecytokines; DAMPs; agonists, DAMPs, monitoring; Molecular PAMPs; alarminsPAMPs, alarmins tools: immunologic studies, pre-clinical studiesNeutrophil Complement, Covalently linked Diagnostics: Immune antibodiesagainst antibodies - IgG, response profiling, CD15, Ly6G or other; IgM,covalent infectious disease antibodies against linkers, spacers,monitoring; neutrophil markers; MHC: antigen vaccination TLR agonists;complexes, monitoring; chronic cytokines; DAMPs; cytokines, TLRinflammatory disease PAMPs; alarmins agonists, DAMPs, monitoring;Molecular PAMPs, alarmins tools: immunologic studies, pre-clinicalstudies Cytokines Antibodies against one Covalently linked Diagnostics:Immune or more epitope of antibodies - IgG, response profiling,cytokine; cytokine IgM, covalent infectious disease binding domain oflinkers, spacers, monitoring; cytokine receptor; PNA vaccination probesagainst cytokine monitoring; chronic gene and/or cytokine inflammatorydisease mRNA; or other monitoring; Molecular tools: immunologic studies,pre-clinical studies

In some examples, the solid phase may be particles ranging from about 2nm in diameter to about 200 mm in diameter and Molecular Nets may beattached to the surface of said particle. Particles may comprisepolystyrene, polyethylene, silica, composite, nylon, PVDF,nitrocellulose, cellulosic, carbon, or other may be magnetic,paramagnetic, fluorescent, barcoded or other.

Molecular Nets may be absorbed or covalently linked to the surface of aparticle in manner to generate pseudorandom or ordered porosities in asingle layer of said Molecular Net or throughout. In its most basicform, a particle may be initially coated with a layer of Molecular Net,which may be connected to a second layer, which may be connected to athird layer. Molecular Net layers may comprise the same capturemolecules at the same or at different concentrations in each layer.Molecular Net particles may also comprise different capture molecules ineach layer and may be fabricated in a manner to incorporate the same ordifferent concentrations of capture molecules compared to previouslayers.

In some examples, Molecular Nets may be attached to a particle surfacein a manner to generate an asymmetric particle having a pre-determinedpolarity. Such a particle may be designed and fabricated with an initiallayer comprising structural molecules with a large diameter, widthand/or length and may be linked to a particle in an asymmetric manner togenerate a polarity. A second layer may be linked to a first layer and athird layer may be connected to a second layer and so on. The number oflayers in a Molecular Net particle may vary depending on use.

In some examples, Molecular Nets may be attached to a segment of aparticle to generate an asymmetric particle having a pre-determinedpolarity. Such a particle is constructed whereby the initial layer iscoated onto a segment of a particle and whereby a second layer isconnected to the initial layer onto the same segment of said particle,and whereby a third layer is connected to the second layer onto the samesegment of said particle, and whereby a fourth layer is connected to thethird layer onto the same segment of said particle.

In some examples, Molecular Nets may be passively absorbed to anonfunctionalized particle surface. In other examples, particle surfacesmay be functionalized and may require activation prior to attachment. Inother examples, particle surfaces may be activated prior tofunctionalization, at which time a Molecular Net may be attached. Yet inother examples, Molecular Nets may be constructed directly on theparticle surface. Attachment of Molecular Nets to a particle may changethe physical and/or chemical features of said particle. In someexamples, Molecular Nets may comprise pseudorandom topological featuresplaced on the surface of a particle. In some examples, Molecular Netparticles may comprise topological features, the topological featurescomprising capture molecules and linkers and may also comprise spacers.Examples of various topological features may include appendages, spikes,plateaus, planes, mounds, fissures, pellicles, stipples, channels, poresand other and may be comprised of capture components directly linkedwithin and/or linked to one or more layer of a Molecular Net.

Other examples of topological features may comprise be pockets, pillars,bumps, branches, projections, ridges, clefts, trellis-like structures,flakes, pellets, spheres, or others. Topological features may bepre-formed in solution and linked to the Molecular Net or may be formedat the time each layer is constructed.

Molecular Nets on particles may comprise heterogeneous capture moleculeswithin one or more layer of a Molecular Net. Benefits of a heterogeneousdesign may relate to the capture of a plurality of analytes having aplurality of surface chemistries on a single particle. Heterogeneouscapture molecules incorporated into a Molecular Net during fabricationmay be randomly distributed throughout each layer; may be stratifiedthroughout each layer; or other, depending on use.

Molecular Nets may be attached to particles to increase surface area ofsaid particle. Molecular Nets may also be used to increase particlediameter. Topological features of a Molecular Net on a particle mayrelate to an increased particle size in addition to analyte capturecapacity.

In some examples, a first layer of Molecular Net may be attached to aparticle surface to modify the physical and/or chemical properties ofparticle. In many commercial particles, “bead effects” or “surfaceeffects” can hamper results and are still not well understood.Traditional conjugation techniques that result in 2D conjugates and 2Dconjugated surfaces often suffer from surface effects. Molecular Netsmay be used to minimize or neutralize bead effects to minimizenon-specific binding to a bead surface, bead autofluorescence, beadinterference with in an assay or other. In some examples, Molecular Netparticles may impart increased analyte binding capacity and may alsoimpart blockade of non-specific binding of undesired analytes toincrease the signal-to-noise ratio in an assay, yield and purity ofpurified analyte or other.

In some examples In yet another aspect, the invention features molecularNet on particle containing more than one layer, wherein each layercontains capture molecules directed against analyte, wherein each layercontains distinct capture molecules directed against distinct analyte,wherein different layers can be directed against different analytes toenable the capture of analyte or a plurality of analytes.

In yet another aspect, a Molecular Net placed on a solid phase surfacemay be used to increase the purity of one or more analyte recovered froma sample. Molecular Net-coated surfaces may reduce non-specific bindingof undesired analyte compared to commercial 2D functionalized surfaces.

In some examples, Molecular Nets placed on particles may significantlyincrease analyte capture capacity of a particle. Additional layering ofa Molecular Net may further increase the number of bound analyte perparticle and may be used to enhance recovery or yield of analyte from asample and may be used to deplete one or more analyte from a sample.

Advantages of using Molecular Nets

Molecular and cellular testing strategies employ the use of single-plexor multi-plex immunoassays, PCR assays, next-generation sequencingtechniques or other to identify the presence of or to measure the amountof one or more analyte in a sample.

In multiplexed assays, reactions may be separated spatially or may becombined into a single testing reaction and may employ solid phasescomprising unique identifiers to provide information. Some examples ofunique identifiers may comprise the use of different barcodes, differentfluorescence emissions, different chemistries, different orderednucleotide tags, or other.

Solid phases may be used in single-plex and multi-plex assays may relyon the specific binding of target analyte to produce a measurable signalor a measurable change in signal and may be used in a direct assay ormay be used in an indirect assay. Measurable signals may be generatedfrom a positive test and may comprise electric, thermal, magnetic,optical, vibrational, isotopic, or other measurable characteristic.

Many of the difficulties in achieving sensitive and reproduciblemeasurements using current strategies result in high non-specificbinding, lower sensitivity, low signal-to-noise and thereby requireupstream sample processing steps to remove as many non-specificcomponents from a sample, coupled with the use of highly sensitivereader technologies and complex algorithms which may be required inorder to determine real signal from the noise, which make them difficultto translate to truly real-time, easy-to-use molecular diagnostics andanalyte measurement tools.

Molecular Nets may be used in place of current commercial approaches andmay generate specific and sensitive analyte capture, detection andmeasurement from a sample. Examples of results obtained from the use ofMolecular Nets in place of current approaches for analyte capture arepresented in FIGS. 1-6. Improvements in assay sensitivity, minimumlevels of analyte detection, and other features may be obtained throughthe use of a Molecular Net in place of current 2D approaches for analytecapture and measurement. Reduction in background noise may be obtainedthrough the use of a Molecular Net in place of current 2D approaches andmay be used to improve analyte purification, analyte purity, and assaysensitivity.

Advantages of a Molecular Net are presented in Table 4 and may include:the rapid capture of one, several or a plurality of molecular andcellular analytes in a raw sample; ability to generate sensitive andspecific signals when used in a test involving indirect and directdetection methods; ability to generate a signal having enhancedfluorescent intensity; ability to concentrate bound analyte; ability tospatially separate bound analyte in a manner that reduces stearichindrance between analytes and/or between detection molecules; enhancedstability; reduced background and others.

TABLE 4 Demonstrated Advantages of Molecular Nets DemonstratedAdvantages Anticipated Impact Finger stick by lancet (~50 uL) Displacesthe need for venipuncture No sample prep - compatible with raw,Displaces the need for sample processing - both unprocessed samplecentrifugation & serum isolation (saves time) Point-of-use testingDisplaces the need and cost of sample transport, with ultimate potentialfor testing at home or in field settings Portability, no complex capitalequipment Displaces the need and cost for off-site CLIA labs Providesimmediate answers (<30 mins) Enables point-of-care treatment and patientmonitoring Multiplexing (multi-analyte analysis) Delivers more robustanswer and eliminates the cost of having to run separate tests persample Simple test procedure Displaces the need for high-complexitytesting (Western blot, Luminex, bead arrays, and PCR) Capable ofproducing simple actionable Enables health care provider (and/orultimately readouts (Binary-No/Yes; Semi- the patient) to make decisionsooner; flexible data quantitative-Low, Mid, High; Fully output enablesnumerous applications quantitative) Stability of test (enzyme-free) =longer Increases shelf life, & reduces costs associated shelf life withstorage issues Cost effective Disposable cassette with the potential formultiple-tests-in-1 High signal, very low noise (demonstratedSignificantly more sensitive than current femtogram range in multipletest types) immunoassaysMolecular Nets and their Use

Molecular Nets may be used in applications where analyte bindingefficiency, analyte binding kinetics, analyte binding capacity, analytedetection, analyte measurement, analyte enrichment, analyte purificationand analyte delivery may be important. Molecular Nets may be used influid phase or may be attached to a solid phase.

Molecular Nets may be attached through absorption or covalent processeson a receptive surface. Examples of solid phases include but are notlimited to nanotubes, metals, particles, microtiter plates, slides,cassettes, probes, lateral flow tests, stents, catheters, valves, bloodtubes, needles, solid phase devices or other. Examples of chemistries ofvarious solid phases that may be compatible for Molecular Net attachmentinclude but are not limited to plastics, other polymer, thin film,colloidal metals, silica, carbon nanotube, protein, carbohydrate, lipid,nucleic acid, cell, tissue or other.

Molecular Nets may be attached to a solid phase device surface tocapture, purify or deplete one or more analyte from a sample. An exampleof using a Molecular Net for analyte capture and/or purification from asample is presented in FIG. 1. Some other examples of Molecular Netsthat may be used to capture and purify analyte from a sample are:Protein A, Protein G or Protein L Net-coated microspheres forimmunoglobulin capture; Streptavidin Net-coated microspheres for biotincapture; TNF Net-coated microspheres for anti-TNF biologic capture; IL6Net-coated microspheres for anti-IL6 biologic capture; IgM Net-coatedmicrospheres for RNA virus capture; Ig Fc Net-coated microspheres forcomplement capture; antigen Net-coated micro spheres forantigen-specific immunoglobulin; antigen-specific immune cell capture;and others. Molecular Nets may be used in chromatography methods for thecapture and purification of one or more analyte from a sample.

Molecular Nets may be used to capture analyte from a sample fordownstream analyte measurement by an independent method, referred toherein as sample prep. Examples of independent methods may include massspectrometry, immunoassay, PCR, next-generation sequencing, qRT-PCR,digital PCR, microscopy, fluorescence, flow cytometry, bead cytometry,or other.

In another aspect, the invention improves signal-to-noise ratios whenused in an assay.

Molecular Nets may be attached to a solid phase device surface tomeasure the presence, absence, modification or concentration of one ormore analyte. Examples of using Molecular Nets for analyte detectionand/or measurement are presented in FIGS. 3 and 4. In some otherexamples, Molecular Nets may be used to simultaneous detect and measure2 or more specific analytes in a direct or indirect manner. Indirectcapture by a Molecular Net may relate to the capture of a primaryanalyte by a specific capture molecule of a Molecular Net that mayenable the detection of one or more related secondary associated withthe captured primary analyte. Molecular Nets may be used as discoverytools capture primary analytes from a sample and enables theidentification, detection or measurement of secondary analytes that arecaptured-by-association. Molecular Nets may be used in this manner fordrug discovery, pathway mapping, and in proteomics, transcriptomics,glycomics, lipidomics, metabolomics, functional genomics, foodomics,nutrition, pharmacology, toxicology and others.

In some examples, Molecular Nets may be used to detect drug resistancein a cell. Cells may be tumor cells, immune cells, microbial cells orother cells. Molecular Nets for these applications may comprise capturemolecules directed against one or more unique features of a cell type.Molecular Nets may additionally be fabricated in a manner to impartsurface topology relating to the capture of in tact cells.

In other examples, Molecular Nets may be fabricated and used in: immunecell reactivity measurement; immune response monitoring; immune responseclassification; immunoglobulin titering; biotinylated molecule capture;multiplex immunoassays; singleplex immunoassays; next-generationsequencing reactions; PCR; microbiome capture; microbiome discovery;mRNA and encoded protein measurement; SNP (single nucleotidepolymorphisms) mapping; SNP detection; disease marker samplepreparation; miRNA capture and/or measurement; post-translationmodification discovery and/or capture and/or measurement; kinaseactivity measurement; or other.

Molecular Nets may have measurable characteristics imparted during thefabrication process and may be used in a direct or indirect manner as asensor. A measurable change in one or more characteristic of a MolecularNet sensor may be detected using commercial approaches employing the useof optical sensing, electrochemical sensing, electromagnetic sensing,electrical impedence, or other. In one example, a Molecular Net sensormay be used to capture and bind an analyte. Analyte binding may resultin a measurable change in a characteristic of a Molecular Net sensor. Abinding event or modifying event pertaining to the Molecular Net sensormay be monitored over a period of time, and the changes in Molecular Netsensor characteristics may be detected, relayed and collected by adevice. Other examples of using a Molecular Net as a sensor may includean analyte binding event, enzymatic reaction, analyte modificationevent, cell differentiation, cell-cell interaction, or other.

Examples of measurable characteristics include but are not limited to:physical shape, height, density, fluorescence intensity, wavelengthshift (FRET or FRAP), vibrational frequency, absorbance, flexibility,refractiveness, conductance, impedence, resistance, melting temperature,denaturation temperature, freezing temperature, and other.

Measuring devices that may be compatible for use with a Molecular Netsensor may comprise: photonic multichannel analyzers, spectrometers,magnetic resonance imagers, magnetic field detectors, optical fibers,glass pipettes, circuits, fluorometers, spectroscopic analyzers, flowcytometers, CCD cameras, microscopes, acoustic chambers, microphones,luminometers, and other. The measuring devices may be used to measurechanges in: thickness, topology, charge, insulation, capacitance,voltage, color, acoustics, vibration, magnetism, enzymatic activity orother characteristics of a Molecular Net used as a sensor.

Additionally, Molecular Nets may be used in flexible circuits, wherebythe capture molecules and/or structural molecules may be connected toconductive molecules. Molecular Net circuits may be used in single-sidedflexible circuits, double access (back bared flex circuits), sculpturedflex circuits, double-sided flex circuits, multilayered flex circuits,ridge flex circuits, ridge-flex boards, polymer thick film flex circuitsor other. Most flexible circuits are passive wiring structures that areused to interconnect electronic components such as integrated circuits,resistor, capacitors and the like, however some are used only for makinginterconnections between other electronic assemblies either directly orby means of connectors. Molecular Nets for use in circuits or for use asa component of a circuit may be comprised of synthetic components or maybe comprised of biochemical capture molecules and/or cells and may befabricated in a manner to be used in a flexible circuit. Molecular Netcircuits may also be used as a sensor.

In some examples, Molecular Net circuits may have specificelectrochemical properties and may be used to monitor various parameterssuch as pH, current, voltage, impedence, or other in anelectrochemical/electrolyte cell. Binding events and modifying eventsthat may occur to Molecular Net may be measurable and may be reflectedby a change in the conductance, current, or voltage. More specifically,the introduction of a sample containing an analyte that has specificbinding affinity for, or is reactive towards, a component in a MolecularNet circuit may be monitored by a change in the electrochemicalproperties of the Molecular Net and/or the surrounding environment.

Examples of binding events on a Molecular Net used in a circuit mayinclude: antibody-antigen interaction, nucleic acid-nucleic acidinteraction, enzyme-substrate interaction, drug-target interaction,enzyme-co-factor interaction, ligand-cell interaction, or any otherspecific surface-chemistry-driven non-covalent interaction. Analytecapture by a Molecular Net may be determined by a change in pH, currentor voltage an electrochemical/electrolyte cell. Measurement of a changein Molecular Net characteristics may also result from one or more, or anaccumulation of modifying events to one or more component in a MolecularNet or to a capture analyte. Examples of modifying events may include:enzyme cleavage; post-translational modification (such asphosphorylation, sulfonation, glycosylation, methylation, or other);removal of a post-translational modification (such asde-phosphorylation); or other similar modification. Modifying events maybe determined by a change in pH, current or voltage in theelectrochemical/electrolyte cell resulting from a change in MolecularNet characteristics or in the surrounding buffer system.

Methods to determine changes in electrochemical properties of aMolecular Net used in a circuit may include the use of scanning ioncurrent microscopy, nanofluidic diodes, nanopores or nanochannels thatdisplay voltage-gated ion current, ion nanogating, nanopore-basedsensing platforms and other methods for measuring the flow, or changesin flow of electrical charge through a medium. More specifically, theinherent sensitivity of many solid-state nanopore sensors is theselective permeability of electrolytes, or ion current, when a bias isapplied across the nanopore. Molecular Nets may be coated onto thesurface of a nanopore and the change in current, voltage, and impedancecan be monitored.

Molecular Net can also be coated onto the surface of a carbon nanotubeand whereby the molecular Net can be constructed in a manner to generatesize exclusion and affinity requirements for analyte sensing.

Bio-Layer Interferometry (BLI) is a label-free technology for measuringbiomolecular interactions. It is an optical analytical technique thatanalyzes the interference pattern of white light reflected from twosurfaces: a layer of immobilized molecular Net on the biosensor tip, andan internal reference layer. Any change in the number of molecules boundto the biosensor tip causes a shift in the interference pattern that canbe measured in real-time. The binding between a ligand immobilized onthe molecular Net-coated biosensor tip and an analyte in solutionproduces an increase in optical thickness at the biosensor tip, whichresults in a wavelength shift, Δλ, which is a direct measure of thechange in thickness of the biological layer. Interactions may bemeasured in real time, providing the ability to monitor bindingspecificity, rates of association and dissociation, or concentration,with precision and accuracy. Only molecules binding to or dissociatingfrom the Molecular Net biosensor may shift the interference pattern andgenerate a response profile. Unbound molecules, may change therefractive index of the surrounding medium, or may change flow rate butwill not affect the interference pattern. This is a uniquecharacteristic of BLI and extends its capability to perform in crudesamples used in applications for analyte—capture molecule binding,quantitation, affinity, and kinetics.

Molecular Net particles may also be used to deliver an active agent.Active agents may be pre-loaded onto capture molecules located in one ormore layer of a Molecular Net. Active agents may comprise: drugs,therapeutics, toxins, viruses, allergens, vaccine components, antigens,immune modulators, surfactants, microbes, oligonucleotides, nutrients,or other. Molecular Net particles may be used in drug or therapeuticdelivery, vaccine delivery, in biofermentation or other. Molecular Netsmay comprise one or more targeting agent on a surface-exposed layer tofacilitate specificity in targeting said Molecular Net particle to aspecific cell type, tissue type, organ type or other. Targeting agentsmay be capture molecules of a Molecular Net. Targeting agents maycomprise: antibodies, receptors, ligands, anti-ligands, or other.Targeting agents in a Molecular Net may be covalently linked to capturemolecules, linkers, and spacers in a surface-exposed layer. Targetingagents may also contribute to the topological features of a MolecularNet.

Examples Example 1. Comparison of Conventional 2D and 3D Molecular NetMicroparticles for Analyte Purification

Molecular Nets comprised of monomeric protein G and linked protein G andcrosslinkers BS³, EMCS, EGS, BMPH and others were used in fabrication.Molecular Net fabrication occurred in real-time on 0.8-10 um magneticpolystyrene microparticle and 45 um nitrocellulose microparticlesurfaces. In some examples, the capture molecule, protein G was used asthe only source of structural support. In some examples, pre-linkedprotein G and monomeric protein G were mixed to serve as additionalstructural support for fabrication of some layers of the Molecular Net.Yet in some other examples, a first layer of Molecular Net comprisedprotein G and Ig Fc region to serve as structural support forfabrication of additional layers of the Molecular Net. In some examples,a protein G Molecular Net comprised 2 layers and in other examples, aprotein G Molecular Net comprised 3 layers. The last layer of theMolecular Net comprised topological features to enhance analyte (in thiscase IgG) binding and recovery from a sample. FIG. 1 is an example ofdata obtained using protein G Molecular Net microparticles in comparisonto commercial protein G microparticles. Briefly, IgG-Alexa 647 wasspiked into human serum (1 ug/tube). Uncoated microparticles, commercialprotein G microparticles and protein G Molecular Net microparticles wereincubated with spiked sample for 15-60 min at RT (at 100,000 particles(uncoated control), 100,000 particles (commercial) and 25,000 particles(Molecular Net). Particles were isolated from samples using magneticseparation and were washed 3x in PBST. Particles were resuspended in2×LSB, boiled and loaded onto an SDS-PAGE. Recovered IgG was measured byCoomassie-stained band densitometry compared to input control. Depictedin FIG. 1 is the percent recovery of input for each purification type.Use of an optimized Molecular Net can reduce background noise in anassay and increase a visible signal. CL Example 2. Effectiveness ofConventional 2D Conjugates for Analyte Detection

Traditional approaches to covalently link capture antibody to a surfaceis a 2-dimensional approach (X and Y planes). as there are no additionallayers added onto the surface of the linked antibodies on a surface.Traditional methods used to covalently link capture antibody to asurface involve a single type of linker, for example EDC, NHS, sulfo-NHSor other. Occasionally, a second linker is used to secure the captureantibody to a surface, but involves removal of the first linker and doesnot add additional height or layering to the antibody-conjugatedsurface. In an example, per manufacturer instructions, anti-humanneuroserpin antibody was coupled to Luminex particles (bead region #54)by linker. Particles were then quenched, blocked, and washed prior touse. Particles were incubated for 15 min in pre-clearedserum+neuroserpin at a concentration range of (0-1 ng/mL). Boundparticles were washed and incubated with biotin-anti-Neuroserpin (10ng/mL) for 15 min. Neuroserpin detection was visualized by avidin-PE (30ng/mL) for 15 min. Washed particles were then analyzed on a Luminex 100,collecting 100 particles per sample. Presented in FIG. 2 is the medianfluorescence intensity (FI) at each dilution above backgroundfluorescence intensity.

FIG. 3. Effectiveness of Molecular Net Microparticles in MeasuringAnalyte.

Molecular Net comprised of identical anti-human neuroserpin antibody (asFIG. 2) and linkers Sulfo-NHS, EMCS, EGS, BMPH and others was fabricatedto provide a 3-dimensional multi-layered (X, Y, and Z planes) matrix.The Molecular Nets were then covalently linked to Luminex microparticles(bead region #54). Assay performance with the 4-layered Molecular Netsare presented in FIG. 3. Improved assay MFI was observed using a3-dimensional multi-layered Molecular Net.

FIG. 4. Effectiveness of a Molecular Net with Topology in MeasuringAnalyte.

Molecular Net comprised of identical anti-human neuroserpin antibody (asFIGS. 2 and 3) and linkers Sulfo-NHS, EMCS, EGS, BMPH and others wasfabricated to provide a 3-dimensional multi-layered (X, Y, and Z planes)matrix. The Molecular Nets were then covalently linked to Luminexmicroparticles (bead region #54). Assay performance with the 5-layeredMolecular Nets are presented in FIG. 4. Improved assay MFI was observedusing a 3-dimensional multi-layered Molecular Net with enhanced topologyin the outer layers.

FIG. 5. Comparison of Traditionally Conjugated Microparticles andMolecular Net microparticles in an ELISA.

Molecular Nets comprised of monoclonal antibody directed againsthuman-TauF and crosslinkers Sulfo-NHS, EMCS, EGS, BMPH and others wereused in fabrication. Molecular Net fabrication occurred in real-time on0.5, 6.3 and 10 um magnetic microparticle surfaces. In some examples,the capture molecule, anti-Tau mAb, was used as the only source ofstructural support. In some examples, the spacer, albumin, was mixedwith the anti-Tau mAb in a first layer at a 1.5:1.0 Molar ratio(albumin:anti-Tau Ab) to serve as additional structural support forfabrication of the first layer. In some examples, a second capturemolecule, human tubulin was used and provided both structural supportand capture roles within a Molecular Net. FIG. 5 is an example of dataobtained using an anti-Tau Molecular Net (LV.P6 Cap-TECH) in comparisonto a commercial Tau microparticle (Partner LV) ELISA (identical assayconditions, identical antibody pair, etc.). FIG. 5 is an example ofusing a Molecular Net to reduce background noise in an assay andincrease a visible signal in an ELISA.

FIG. 6. Comparison of Traditionally Conjugated Microparticles andMolecular Net Microparticles Using Luminex.

Molecular Net comprised of monoclonal antibody directed againsthuman-thyroid stimulating hormone and crosslinkers EDC, BS(PEG)₉, EMCS,EGS, BMPH and others were used in fabrication. Molecular Net fabricationoccurred in real-time on Luminex magnetic microparticle surfaces. Insome examples, the capture molecule, anti-TSH mAb, was used as the onlysource of structural support. In some examples, the spacers, PEG,heat-denatured lysozyme and others were mixed with the anti-TSH mAb in afirst layer at a 1.0:2.0 Molar ratio (spacer:anti-TSH Ab) to serve asadditional structural support for fabrication of the first layer. Insome examples, an anti-TSH Molecular Net comprised 4 layers and in otherexamples, an anti-TSH Molecular Net comprised 6 layers with the lastlayer comprising topological features to enhance analyte binding andperformance in a Luminex. FIG. 6A is an example of using a

Molecular Net to increase the overall MFI in a Luminex assay. FIG. 6B isexemplary data obtained in a Luminex assay to increase the minimumlevels of detection in a Luminex assay.

FIG. 7. Exemplary Molecular Nets on Particles.

FIG. 7 depicts some examples in which Molecular Nets may be placed ontoa particle surface. In some examples, a Molecular Net is placed on aparticle surface (FIG. 7A, 1001) in a circumferential manner whereby aMolecular Net having X, Y and Z spatial orientation may be fairlysymmetrical and where each layer (examples of 3 layers, 1002, 1003 and1004) adds to the Z plane of the particle. In some examples, MolecularNets may be placed onto a particle surface (FIG. 7B, 1005) in anasymmetrical manner whereby a Molecular Net having X, Y and Z spatialorientation may be placed onto the surface of a particle in a manner togenerate a polarity of the particle and where each layer (examples of 3layers, 1006, 1007 and 1008) adds to the Z plane of the particle in alayer-dependent manner (for example, some layers may have less heightand other layers may have more height). In other examples, MolecularNets may be placed onto a portion of particle surface (FIG. 7C, 1009) inan asymmetrical manner whereby a Molecular Net having X, Y and Z spatialorientation may be placed onto the surface of a particle in a manner togenerate a polarity of the particle and where each layer (examples of 3layers, 1010, 1011 and 1012) adds to the Z plane of the particle in alayer-dependent manner (for example, some layers may have specificityfor an analyte and other layers may have specificity for otheranalytes).

FIG. 8. Exemplary Molecular Net Topological Features on Particles.

FIG. 8 depicts some examples in which Molecular Nets may be placed ontoa particle surface. In some examples, a Molecular Net is placed on aparticle surface (FIG. 8A, 2001) in a circumferential manner whereby aMolecular Net having X, Y and Z spatial orientation may be fairlysymmetrical in one layer (2002) and where each layer (examples of 3layers, 2002, 2003 and 2004) adds to the Z plane of the particle indiffering and asymmetric ways (for example, topology is generated). Insome examples, Molecular Nets may be placed onto a particle surface(FIG. 8B, 2005) in an asymmetrical manner whereby a Molecular Net havingX, Y and Z spatial orientation may be placed onto the surface of aparticle in a manner to generate structural features (2008) throughoutlayers 1 (2006), 2 (2007) and 3 (2008) of the particle and where eachlayer adds to the Z plane of the particle in a layer-dependent manner(for example, some layers may have less height and other layers may havemore height). Additionally, the structural elements in each layer mayalso serve an analyte capture role in a Molecular Net. In otherexamples, Molecular Nets may be placed onto a portion of particlesurface (FIG. 8C, 1009) in an asymmetrical manner whereby a MolecularNet having X, Y and Z spatial orientation may be placed onto the surfaceof a particle in a manner to generate a polarity of the particle andwhere each layer (examples of 4 layers, 2010, 2011, 2012 and 2013) addsto the Z plane of the particle in a layer-dependent manner and wherebyeach layer may serve both structural and analyte capture roles. Forexample, some layers may have specificity for an analyte based on size(e.g., FIG. 8C, 2010) and outer layers (e.g., FIG. 8C, 2013) may havespecificity for analyte of a larger size.

FIG. 9. Exemplary Molecular Nets for Analyte Delivery.

FIG. 9 depicts some examples in which Molecular Nets may be placed ontoa particle surface for use in analyte capture or targeted analytedelivery. In some examples, a Molecular Net is placed on a particlesurface (FIG. 9A, 3001) in a circumferential manner whereby a MolecularNet having X, Y and Z spatial orientation may be fairly symmetrical inone layer (3002) and where each layer (examples of 3 layers, 3002, 3003and 3004) adds to the Z plane of the particle in differing andasymmetric ways (for example, topology is generated). In some examples,analyte cargo (3003) may be pre-loaded onto capture molecules in one ormore layer of a Molecular Net. In outer layers, different capturemolecules may be linked into a Molecular Net to generate a topologyand/or an affinity for a different target analyte. In some examples, apre-loaded analyte may comprise a drug, a therapeutic, siRNA, miRNA,dsRNA, virus, toxin, immunogen or other. Pre-loaded cargo may benon-covalently associated with one or more type of capture molecule in alayer of a Molecular Net. In some examples, different capture molecules(3004) of a Molecular Net may be arranged in the outer layers of aMolecular Net and may serve topological and analyte capture roles. Thedifferent capture molecules may have specificity for one or moredifferent analyte, the analyte of which may comprise an antibody, ananti-ligand, a ligand, a receptor, an antigen or other and may serve oneor more structural and/or affinity and/or targeting role.

In some examples, Molecular Nets may be placed onto a particle surface(FIG. 9B, 3005) in a manner whereby analyte cargo (3006) may bepre-loaded in all layers of a Molecular Net. In some layers, capturemolecules may be used to generate topological features (3008) that serveparticle-targeting roles. In some examples, outer layers of a MolecularNet particle may target said particle to a specific cell, tissue, organor other.

FIG. 10. Molecular Nets for Analyte Purification from a Sample

Molecular Nets may be used in sample purification processes (example isprovided in FIG. 10). In some examples, Molecular Net designed andfabricated to deplete one or more analyte may be used to treat a samplefor analyte depletion. Exemplary methods may include incubation ofMolecular Net with sample for about 15 minutes to about 24 hours in abatch slurry or in a chromatography column. Sample supernatant orflowthrough may be collected depending on preferred method. MolecularNets may be collected and analyzed using various methods for thepresence and amount of captured analyte. Molecular Net-treated samplemay be collected and analyzed using various methods to determine theresidual presence of analyte in the sample or may be analyzed for otheranalytes in the sample.

FIG. 11. Molecular Nets for Analyte Detection & Measurement from aSample

Molecular Nets may be used in an analyte measurement tool or adiagnostic tool (example is provided in FIG. 11). In some examples,Molecular Net designed and fabricated to capture one or more analyte maybe used to treat a sample for analyte detection and measurement.Exemplary methods may include incubation of Molecular Net with samplefor about 15 minutes to about 2 hours in a batch slurry, cassette,slide, microtiter plate or other. Sample supernatant or flowthrough maybe collected depending on preferred method. Molecular Nets may becollected and analyzed using various methods for the presence and amountof captured analyte(s). Molecular Net-treated sample may also becollected and analyzed using various methods to measure other analytes.Methods for measuring changes in Molecular Net characteristics mayinclude optical, electrophoretic, electrical, magnetic, chemical,thermal or other.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes can be made and equivalents can besubstituted without departing from the scope of the invention. Inaddition, many modifications can be made to adapt a particularsituation, material, composition of matter, process, process step orsteps, to achieve the benefits provided by the present invention withoutdeparting from the scope of the present invention. All such modificationare intended to be within the scope of the claims appended hereto.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an indication that any such document is pertinent prior art, nor doesit constitute any admission as to the contents or date of the same.

1-52. (canceled)
 53. A method of manufacturing a device for capturing an analyte, the method comprising: providing a solid phase; and placing a molecular net on at least a portion of a surface of the solid phase, the molecular net including capture molecules of at least one type coupled to each other by linker molecules of a plurality of types to form a covalently-linked multi-layered three-dimensional matrix, the capture molecules configured to bind to the analyte.
 54. The method of claim 53, wherein placing a molecular net on includes pre-fabricating the molecular net and absorbing the molecular net to the surface of the solid phase.
 55. The method of claim 53, wherein placing a molecular net on includes covalently linking the molecular net to the surface of the solid phase.
 56. The method of claim 53, wherein placing a molecular net on includes constructing the molecular net directly on the surface of the solid phase.
 57. A composition comprising: (a) a solid phase; (b) a molecular net capable of binding to at least one analyte and coupled to at least a portion of a surface of the solid phase, the molecular net comprising more than one layer, which more than one layers comprise at least a first layer and a second layer, and each of the layers comprises capture molecules capable of specifically binding to the analyte(s); and, (c) at least two different linker molecules, wherein: the capture molecules are coupled to each other within each layer by the at least two different linker molecules, the molecular net is an internally covalently-linked three dimensional matrix, the capture molecules within each layer can be of the same or different types, the capture molecules of one layer can be of the same or different types as the capture molecules of another layer, and the capture molecules comprise one or more antibodies directed to the analyte.
 58. The composition of claim 57, wherein the molecular net is pre-fabricated before coupling to the solid phase.
 59. The composition of claim 57, wherein the molecular net is covalently linked to the surface of the solid phase.
 60. The composition of claim 57, wherein the molecular net is constructed directly on the surface of the solid phase.
 61. A method of measuring a quantity of an analyte in a sample, the method comprising: providing one or more devices each comprising a solid phase and a molecular net covering at least a portion of a surface of the solid phase, the molecular net including capture molecules of at least one type coupled to each other by linker molecules of a plurality of types to form a covalently-linked multi-layered three-dimensional matrix, the capture molecules configured to bind to the analyte; exposing the devices to the sample; and allowing at least a portion of the analyte to bind to the capture molecules of the molecular nets of the devices.
 62. The method of claim 61, further comprising measuring a change in the sample. 